14-3-3 proteins promote synaptic localization of N-methyl d-aspartate receptors (NMDARs) in mouse hippocampal and cortical neurons

One of the core pathogenic mechanisms for schizophrenia is believed to be dysfunction in glutamatergic synaptic transmissions, particularly hypofunction of N-methyl d-aspartate receptors (NMDARs). Previously we showed that 14-3-3 functional knockout mice exhibit schizophrenia-associated behaviors accompanied by reduced synaptic NMDARs in forebrain excitatory neurons. To investigate how 14-3-3 proteins regulate synaptic localization of NMDARs, here we examined changes in levels of synaptic NMDARs upon 14-3-3 inhibition in primary neurons. Expression of 14-3-3 protein inhibitor (difopein) in primary glutamatergic cortical and hippocampal neurons resulted in lower number of synaptic puncta containing NMDARs, including the GluN1, GluN2A, or GluN2B subunits. In heterologous cells, 14-3-3 proteins enhanced surface expression of these NMDAR subunits. Furthermore, we identified that 14-3-3ζ and ε isoforms interact with NMDARs via binding to GluN2A and GluN2B subunits. Taken together, our results demonstrate that 14-3-3 proteins play a critical role in NMDAR synaptic trafficking by promoting surface delivery of NMDAR subunits GluN1, GluN2A, and GluN2B. As NMDAR hypofunctionality is known to act as a convergence point for progression of symptoms of schizophrenia, further studies on these signaling pathways may help understand how dysfunction of 14-3-3 proteins can cause NMDAR hypofunctionality and lead to schizophrenia-associated behaviors.


Introduction
N-methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play a central role in excitatory synaptic transmission and plasticity [1,2]. They form heteromeric assemblies composed of GluN1, GluN2, and GluN3 subunits, which contain a large amino (N)-terminal extracellular domain, three membrane-spanning domains, a 'hairpin' loop that forms the pore-lining region, and an intracellular carboxy (C)-terminal domain [2]. NMDARs have a voltage-sensitive Mg 2+ block and high permeability to Ca 2+ . This Ca 2+ influx triggers signal transduction cascades that regulate synaptogenesis, experience-dependent synaptic remodeling, and long-lasting changes in synaptic efficacy such as long-term potentiation and long-term depression [1,3,4]. NMDARs are, therefore, essential for cognition, memory, and length rat GluN2C plasmid was kindly provided by Dr. Katherine Roche at the National Institutes of Health (NIH). pEGFP-GluN2A (Plasmid #17924) and pEGFP-GluN2B (Plasmid #17925) were purchased from Addgene (Cambridge, MA). The pSCM138 plasmid, expressing for enhanced YFP-fused doublet of R18 peptide (also referred to as difopein), was kindly provided by Dr. Haian Fu at Emory University. Full-length human 14-3-3z in the mammalian expression vector pcDNA3.1 was tagged with HA between EcoRI-XbaI sites using the following primers: (1) forward primer: CCAGGTGAATTCCACAAAATGGATAAAATGAGCTGGTTC; (2) reverse primer: CTTGCTTCTAGACTAAGCGTAGTCTGGGACGTCGTATGGGTAATTTTCC CCTCCTTCTCCTGC. Full-length human 14-3-3ε in the mammalian expression vector pcDNA3.1 was tagged with HA between BamHI-XhoI sites using the following primers: (1) forward primer: GAACCCTGGATCCCACAAAATGGATGATCGAGAGGATCTG; (2) reverse primer: CTTGCTCTCGAGAGTAGCGTAGTCTGGGACGTCGTATGGGTAGACTAAAAGCAGAA GGTG CAG. All cDNA constructs used were verified by DNA sequencing.

Virus construction
All AAV used here were constructed and produced by the Obio Technology (Shanghai) CO., LTD as previously described [37]. To construct AAV-CaMKIIα-YFP and AAV-CaMKIIα-YFP-difopein, the cDNA encoding either YFP or YFP-difopein was subcloned into the recombinant AAV vector to produce either pAOV-CAMKIIα-YFP or pAOV-CaMKIIα-YFP-difopein respectively. These viruses (AAV serotype 2/9) were then produced using the triple transfection method in human embryonic kidney 293 (HEK293) cells and AAV titers were determined by real-time PCR.

Primary culture of cortical and hippocampal neurons
All animal procedures were carried out in accordance with the guidelines of Florida State University's Institutional Animal Care and Use Committee (protocol #: 20190006) and all studies were performed in accordance with the recommendations in the NIH's Guide for the Care and Use of Laboratory Animals. All mice were housed in a humidity-and temperature-controlled room maintained on a 12:12 h light: dark cycle and provided ad libitum access to standard rodent chow and water. Euthanasia of individual fetuses were conducted through decapitation with surgical scissors. Cultures of primary cortical and hippocampal neurons were prepared using a standard procedure as previously described, with minor modifications [38]. Briefly, cerebral cortices and hippocampi from five to six postnatal day 0 C57/BL6J wildtype mice (Jackson Laboratories, Stock #000664) of either sex were dissected with the aid of a stereo microscope. Isolated cerebral cortices and hippocampi were digested with papain (Worthington Biochemical Corporation, Cat: LK003176) for 5 minutes at 37˚C. Isolated neurons were seeded on Poly-L-Lysine (Sigma, Cat: P4832) coated plates at a density of 470 cells/ mm 2 ) software. The selected cell was imaged in serial optical sections at 0.13 μm intervals over a total depth of 2.5 μm, for a total of 19 optical sections. Maximum intensity projections were generated from these sections using Zen Blue (Carl Zeiss) software. All other image processing was performed using ImageJ (NIH) software. The experimenter was blinded to condition and YFP+ dendritic region of interest (ROI) (50 μm × 10 μm) was selected at random for analysis. Numbers of NMDAR synaptic puncta as defined by colocalization of NMDAR subunit with SYP were quantified in the selected ROI using the Puncta Analyzer program as described previously [39]. Averages from three dendrites per cell (total of 21-27 dendrites per condition) were calculated and a total of 7-9 cells from three different coverslips was analyzed for each experimental condition.

Immunocytochemistry assay in tsA201 cells
Surface and total immunofluorescence staining of EGFP-GluN2A and EGFP-GluN2B in tsA201 cells was performed as described previously [38] with minor modifications. Briefly, twenty-four hours post-transfection, cells were fixed at 37⁰C for 10 minutes with 4% paraformaldehyde and 4% sucrose in PBS, pH 7.4. The fixed cells were blocked with 5% goat serum in PBS for 30 minutes at room temperature. Cells were stained using mouse monoclonal anti-GFP antibody (Santacruz, Cat: sc-9996, AB_627695, 1:50 dil.) in PBS containing 5% goat serum with gentle shaking overnight at 4⁰C to label surface proteins and were probed with Alexa Fluor 594-conjugated donkey anti-mouse IgG (H+L) (Molecular Probes, Cat: A-21203, AB_141633, 1:1000 dil.) secondary antibody for 1 hour at room temperature. Following incubation with secondary antibody, cells were washed with 1x PBS for 5 minutes at room temperature, and mounted on slides with Vectashield antifade mounting medium (Vector Labs, Cat: H-1000). Fluorescence images were acquired using a Zeiss LSM-510 Meta confocal microscope (Zeiss, Germany) with a 20 x 0.8 NA lens in the inverted position, performed with identical gain, contrast, laser excitation, pinhole aperture, and laser scanning speed. Blinded to condition, a ROI was selected at random from where the center of the camera field was oriented to capture digital images of fluorescence emissions at 488 nm and 594 nm using Zen Black (Carl Zeiss) software. Twelve-bit tiff files were exported using Zen Blue (Carl Zeiss) software and all other image processing was performed using ImageJ (NIH). Fluorescence intensity was quantified as described previously [38]. Cells were selected at random from each image using the ROI tool (25 μm × 25 μm square) and intensity from the A594 (surface) and A488 (total) channels was quantified using ImageJ. The ratio of cell surface to total channel expression as a measure of intensity (surface expression over surface plus intracellular expression), representing either membrane: total EGFP-GluN2A or membrane: total EGFP-GluN2B, allowed comparisons between various conditions assayed from different batches of cells. A total of 12 cells from three different coverslips were analyzed for each experimental condition.

Coimmunoprecipitation and western blot
Western blotting and coIP assays were performed as described previously with minor modifications [41]. tsA201 cells were transiently transfected with GluN1-1a and GluN2A or GluN2B with pcDNA (vector control), HA-14-3-3z, or HA-14-3-3ε. Forty-eight hours post-transfection, cells were resuspended in cold lysis buffer containing 1% Triton X-100, 10 mM EDTA, 120 mM NaCl, 50 mM KCl, 50 mM NaF, 2 mM DTT, and protease inhibitors. After removing insoluble debris by centrifugation, the clear lysate was incubated with mouse monoclonal anti-HA antibody (Santacruz, Cat: sc-7392-X, AB_627809, 1-2 μg) for 2 hours at 4⁰C while rotating and the immunocomplex was precipitated with Protein A/G PLUS-Agarose beads (Santacruz, Cat: sc-2003, AB_10201400) overnight at 4⁰C while rotating. The beads were subsequently washed three times with lysis buffer and then boiled for 5 minutes at 95⁰C in 2 x SDS sample buffer and were further analyzed by immunoblotting. Proteins in cell lysate or IP were separated on 10% polyacrylamide gels and transferred to 0.45 μm nitrocellulose membranes (Biorad, Cat: 1620115). After blocking with 5% nonfat milk in PBST (0.1% Tween-20), the blots were probed with appropriate primary antibodies in blocking buffer while shaking overnight at 4⁰C. Similar primary antibodies were used, western blotting and data acquisition was performed as described for the surface biotinylation assay. The mouse hippocampal brain lysates were prepared as previously described [34,35,37]. For coimmunoprecipitation from mouse brain lysates, dissected hippocampi from three adult C57/BL6J wild-type mice (~80mg) were homogenized in 1.0 ml ice-cold radioimmunoprecipitation (RIPA) buffer (50mM Tris-HCl, pH 8.0, 150mM NaCl, 1% [vol/vol] NP40, 0.5% [mass/ vol] sodium deoxycholate, 5mM NaF, 1mM Na3VO4, 1mM EDTA, 1mM EGTA, 1mM PMSF) with a micro-pestle. Brain lysates upon centrifugation were mixed with protein A/G PLUS-Agarose beads (Santacruz, Cat: sc-2003, AB_10201400) and incubated for 2 hours at 4˚C while rotating. The pre-cleared brain lysates were then transferred to a new tube and beads were discarded. Supernatants were then incubated either with rabbit polyclonal anti-14-3-3z antibody (1-2 μg) or rabbit polyclonal anti-mCherry (rabbit IgG) as a negative control (Rockland, Cat: 600-401-P16, 1-2 μg) overnight at 4⁰C while rotating. The rabbit polyclonal anti-14-3-3z antibody was generated from our lab as described previously [42]. The immunocomplex was then precipitated with Protein A/G PLUS-Agarose beads (Santacruz, Cat: sc-2003, AB_10201400) for 2 hours at 4⁰C while rotating. The beads were subsequently washed six times with RIPA buffer containing 0.2% SDS, spinning down beads in-between washes at 1000 x g for 3 min at 4⁰C. After the final wash, supernatant was discarded and beads were boiled for 5 minutes at 95⁰C in 2 x SDS sample buffer. Proteins in hippocampal brain lysate or IP were separated on 8 or 10% polyacrylamide gels and transferred to 0.45 μm nitrocellulose membranes (Biorad, Cat: 1620115). Western blotting and data acquisition were performed similarly as described for coIP on tsA201 cells.

Statistical analyses
Statistical analysis was performed using a two-tailed unpaired Student's t-test for significance for the quantitative analysis for synaptic localization of primary neurons. One-way repeatedmeasures of variance (ANOVA) with Bonferroni's multiple comparisons test were used for all tsA201 cell results. GraphPad Prism 9.0 (San Diego, CA) was used to analyze all data. Outliers were excluded from the data sets and were detected using the robust regression and outlier removal method set to an average false discovery rate of less than 1%. A value of � p < 0.05 was considered to be a statistically significant difference. All data are presented as means ± standard deviation (SD) and values of n are indicated in the respective figure.

14-3-3 proteins enhance NMDAR synaptic localization in glutamatergic cortical and hippocampal neurons
To investigate the role of 14-3-3 proteins in synaptic localization of NMDARs, we assessed the effect of 14-3-3 inhibition on synaptic levels of NMDARs in primary glutamatergic cortical and hippocampal neurons. In this experiment, neurons were infected with the Ca 2+ /calmodulin-dependent protein kinase II α (CAMKIIα) promoter-driven adeno-associated virus (AAV) that expresses either YFPfused difopein or YFP (control). Difopein is an established 14-3-3 inhibitor that binds to 14-3-3 proteins with high affinity, thereby competitively inhibits 14-3-3 ligand interactions [43]. Synaptic localization of NMDARs was assessed by quantifying numbers of GluN1, GluN2A, and GluN2B containing puncta that were colocalized with a synaptic marker synaptophysin I (SYP). In neurons infected with AAV-YFP difopein, numbers of synaptic GluN1 puncta (per 50 μm) were decreased in both cortical and hippocampal neurons compared to that in neurons infected with AAV-YFP (Fig 1A and 1B). Similarly, infection of AAV-YFP difopein reduced the numbers of synaptic GluN2A puncta in both cortical and hippocampal neurons (Fig 2A and 2B). However, AAV-YFP difopein infected neurons showed decreased numbers of synaptic GluN2B puncta only in hippocampal neurons and showed no changes in numbers of synaptic GluN2B puncta in cortical neurons (Fig 3A and 3B). Together, these data demonstrate that 14-3-3 protein inhibition leads to a reduction of synaptic localization of NMDARs in primary glutamatergic cortical and hippocampal neurons.

14-3-3 proteins promote surface expression of GluN1, GluN2A, and GluN2B subunits in tsA201 cells as assessed by surface biotinylation assay
To understand how 14-3-3 proteins might enhance synaptic localization of NMDARs, we asked whether 14-3-3 proteins can promote surface expression of NMDARs, which involves receptor subunit assembly in the ER, followed by ER exit [13]. Deregulation of surface expression of NMDARs can profoundly disrupt their synaptic targeting [7]. Previously, others reported that 14-3-3z and ε play a significant role in promoting cell surface delivery of a number of ion channels and receptors including the GluN2C subunit [44][45][46][47][48][49][50][51][52][53]. Thus, we first performed surface biotinylation experiments in heterologous cells to assess the level of receptors expressed in the plasma membrane. In these experiments, we transiently co-expressed GluN1-1a subunit with GluN2A or GluN2B in tsA201 cells. To assess the effects of 14-3-3 proteins, we co-transfected cDNAs that encode for either difopein (pSCM138), hemagglutinin (HA) tagged 14-3-3z or 14-3-3ε, or vector control (pcDNA3). We then performed surface biotinylation assay and used western blotting to quantify surface and total expression levels of NMDARs. When GluN1-1a was co-expressed with GluN2A, the ratios of surface to total expression of GluN1 and GluN2A were decreased with 14-3-3 inhibition. In addition, the ratio of GluN1 surface to total expression increased with 14-3-3z and ε overexpression, while GluN2A expression only increased with the overexpression of 14-3-3z (Fig 4A-4C). Similarly, in cells co-expressing both GluN1-1a and GluN2B, the ratio of surface to total expression of GluN2B was reduced by co-transfection of difopein and increased by overexpression of both 14-3-3z and 14-3-3ε, while increased GluN1 surface expression was observed only with 14-3-3z overexpression (Fig 4D-4F). Collectively, our results demonstrate that 14-3-3 proteins positively enhance surface expression of NMDARs in heterologous cells.

14-3-3 proteins promote surface expression of functional GluN2A and GluN2B subunits in tsA201 cells as examined by immunocytochemistry
To further verify the role of 14-3-3 proteins on enhancing surface expression of NMDARs, we examined surface levels of NMDARs using immunocytochemistry (ICC). In this set of

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The role of 14-3-3 proteins in synaptic localization of NMDARs

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The role of 14-3-3 proteins in synaptic localization of NMDARs experiments, we co-transfected GluN1-1a in tsA201 cells with GluN2A or GluN2B subunits that are N-terminally fused with enhanced GFP (EGFP-GluN2A or EGFP-GluN2B). The effect of 14-3-3 proteins on surface expression of GluN2A and GluN2B was assessed either by co-

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The role of 14-3-3 proteins in synaptic localization of NMDARs expression of exogenous 14-3-3z or 14-3-3ε proteins, or through application of a small molecule 14-3-3 inhibitor BV02, which unlike the pSCM138 plasmid, does not by itself emit GFP signal. The surface expressed EGFP-GluN2A or GluN2B subunits were labeled with primary anti-GFP antibody followed by red (shown in red) fluorophore-conjugated secondary antibodies in unpermeated cells, while their total expressions were visualized by direct EGFP signals (Fig 5). We then measured fluorescence intensity of both red (surface) and green (total) channels in transfected tsA201 cells to determine the ratio of surface to total expression for EGFP-

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The role of 14-3-3 proteins in synaptic localization of NMDARs tagged GluN2A and GluN2B. Consistent with our surface biotinylation findings, we found that the ratio of surface to total expression for EGFP-tagged GluN2A was decreased in the presence of 14-3-3 inhibitor compound (BV02) and increased only when 14-3-3ε was overexpressed (Fig 5A and 5B). Similarly, the ratio of surface to total expression for EGFP-tagged GluN2B was decreased when 14-3-3 inhibitor compound was administered, although there were no significant changes in surface expression when 14-3-3z or ε was overexpressed ( Fig  5C and 5D). These results, therefore, confirmed that 14-3-3 can positively promote surface expression of functional GluN2A and GluN2B subunits in heterologous cells.

Discussion
Here we demonstrate that inhibition of 14-3-3 proteins leads to decreased synaptic localization of NMDAR subunits GluN1, GluN2A, and GluN2B in primary cultures of glutamatergic cortical and hippocampal neurons. In addition, results from both surface biotinylation and ICC show that 14-3-3 proteins enhance surface expression of these NMDAR subunits in heterologous cells. These results are consistent with our previous reports, which show that 14-3-3 FKO mice display NMDAR hypofunctionality as exhibited by reduced protein levels of GluN1 and GluN2A in hippocampal PSD fractions and decreased NMDAR-mediated excitatory postsynaptic currents at the hippocampal CA3-CA1 synapse [35]. Inhibition of 14-3-3 proteins in both primary glutamatergic cortical and hippocampal neurons resulted in decreased numbers of synaptic puncta in GluN1-and GluN2A-containing NMDARs (Figs 1A, 1B, 2A and 2B). Interestingly, the numbers of synaptic puncta of GluN2B-containing NMDARs decreased significantly only in glutamatergic hippocampal neurons (Fig 3A and 3B). However, we did observe that 14-3-3 interacts and modulates both GluN2A-and GluN2B-containing NMDARs in our in vitro and in vivo experiments (Figs 4-6 and S1 Fig). It is likely that 14-3-3 proteins play a region-specific role in regulating synaptic localization of certain subtypes of NMDARs in neurons. Indeed, we have previously reported that acute inhibition of 14-3-3 proteins in the hippocampal glutamatergic neurons, but not in the prefrontal cortical glutamatergic neurons, of wild-type mice using AAV-delivered difopein is sufficient to induce SCZ-associated behavioral deficits and reduce protein levels of GluN1 and GluN2A in the PSD fractions [37]. Further studies are certainly needed to help determine the region-specific and subtype-specific differences on the role of 14-3-3 proteins in the brain. Taken together, our findings suggest a Fig 6. 14-3-3z and ε interact with GluN2A-and GluN2B-containing NMDARs. tsA201 cells co-expressing NMDAR subunits and HA-tagged 14-3-3z or ε isoforms were lysed and proteins were immunoprecipitated (IP) with anti-HA antibodies. Input and IP protein samples were separated by SDS/PAGE and probed with NMDAR subunit antibodies. As controls, cell lysates expressing vector control (pcDNA) were used for comparison and all immunoblots were reprobed with anti-HA and anti-GAPDH antibodies. Images shown are representative western blots from three independent experiments. IB, antibody used for immunoblot analysis. A, When GluN1-1a is transiently expressed alone, neither 14-3-3z nor ε is coimmunoprecipitated from cells expressing either of these 14-3-3 isoforms as compared to cells expressing pcDNA (vector control). � There is a non-specific band detected~115kDa when probed with anti-GluN1 antibody. B, 14-3-3z and ε coimmunoprecipitate both GluN1 and GluN2A subunits from cells expressing functional GluN2A subunits, which do not coimmunoprecipitate from cells only expressing pcDNA. C, Cells expressing functional GluN2B subunits result in coimmunoprecipitation of both GluN1 and GluN2B subunits from cells expressing 14-3-3z or ε isoform, which does not result from cells expressing only pcDNA. https://doi.org/10.1371/journal.pone.0261791.g006

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link between NMDAR hypofunctionality and SCZ-associated behaviors. The purpose of this study is to understand how 14-3-3 proteins regulate NMDAR functions by modulating their synaptic localization.
The trafficking of NMDARs to synapses involves three critical steps including subunit assembly and processing, dendritic transport, and insertion onto synapses [13,14]. In the ER, NMDAR subunits undergo a quality control mechanism to ensure that only properly folded and assembled subunits undergo surface delivery. This entails the masking of ER retention signals located in different regions of GluN subunits either by forming functional NMDAR subunits and/or interaction with other proteins [56][57][58][59]. A number of studies have shown that 14-3-3 proteins can facilitate efficient cell surface expression of several membrane proteins including ion channels and receptors by interacting with these proteins [46,55,[60][61][62]. Here we show that 14-3-3 proteins enhance surface expression of GluN1, GluN2A, and GluN2B subunits as assessed using surface biotinylation and ICC in heterologous cells. 14-3-3 inhibition using an isoform-independent 14-3-3 inhibitor decreased surface expression of GluN1, GluN2A, and GluN2B. In contrast, increasing exogenous levels of 14-3-3z or ε did not consistently result in a significant effect, although there were trends towards an increased surface expression of NMDARs (Figs 4A-4F and 5A-5D). We chose to assess specific functions of 14-3-3z and ε isoforms based on their established role as key regulators in a number of neurodevelopmental processes and their association with neurological disorders, including schizophrenia [63]. However, since endogenous 14-3-3 proteins are abundantly expressed in heterologous cell lines, controlling for variability that results from transient transfection and achieving high enough exogenous 14-3-3 isoform expression level poses a significant challenge. Future studies using isoform-specific inhibitors may help determine the effects on surface expression and synaptic localization of NMDARs. In addition, increasing sample sizes may help reduce variability in experiments involving 14-3-3 overexpression. Nevertheless, our findings indicate that 14-3-3 proteins may enhance synaptic localization of NMDARs by playing a positive role in surface delivery of GluN subunits. Future studies will help us further determine whether 14-3-3 can affect synaptic localization of NMDARs through its involvement in other steps including dendritic transport and synaptic insertion.
Previous studies have shown that 14-3-3 proteins regulate forward trafficking and enhance synaptic localization of GluN2C in cerebellar granule neurons. These studies also found that all 14-3-3 isoforms except 14-3-3σ bind to GluN2C via a motif containing phosphorylated serine 1096 located at the C-terminal domain. They further demonstrated that phosphorylationdependent protein-protein interaction between 14-3-3 and GluN2C is crucial for forward transport and synaptic localization of GluN2C-containing NMDARs [44,60]. In this study, we determined that 14-3-3 proteins interact with GluN2A-and GluN2B-containing NMDARs in both co-transfected tsA201 cells and hippocampal mouse brain lysates (Fig 6A-6C and S1  Fig). 14-3-3 proteins may play a similar role in enhancing forward transport and synaptic localization of GluN2A and GluN2B subunits through direct binding. Here we identified S1291 and S1312 for GluN2A and S1303 and S1323 for GluN2B as potential candidates of 14-3-3 binding sites for GluN2A (UniProt ID: Q00959) and GluN2B (UniProt ID: Q00960) subunits using the 14-3-3-Pred webserver [64] (S2A Fig). Previously, others also reported that GluN2A S1291 is phosphorylated by protein kinase C and this phosphorylation potentiates GluN2A-containing receptor currents [65,66]. In addition, GluN2B-mediated currents are enhanced following phosphorylation of GluN2B S1303 by protein kinase C, death-associated protein kinase 1, and CAMKII [67][68][69][70][71][72]. Based on these predictions and knowing that 14-3-3 interacts with target proteins via phosphoserine or phosphothreonine binding motifs [25], we then generated GluN2A S1291A/S1312A and GluN2B S1303A/S1323A mutants using sitedirected mutagenesis. We then performed coIP using these double mutants to assess their binding with 14-3-3 proteins. Based on our preliminary findings, however, mutations to these two serine residues on either GluN2A or GluN2B subunit did not significantly reduce binding of 14-3-3 proteins to these subunits (S2B and S2C Fig). It is likely that there may be other 14-3-3 binding motifs on GluN2A and GluN2B that play a critical role in protein-protein interactions between 14-3-3 proteins and GluN2A and GluN2B subunits. Further identifying kinases that regulate 14-3-3 binding to functional GluN2A and GluN2B subunits may point us to specific 14-3-3 binding sites on GluN2A and GluN2B subunits. Considering that difopein can inhibit 14-3-3 binding to a number of proteins in neurons, 14-3-3 proteins may indirectly affect synaptic localization and surface expression of NMDARs through their interactions with other proteins. Thus, future studies are needed to further determine the molecular details of the 14-3-3/NMDAR complex. This may help generate 14-3-3 binding-deficient NMDAR mutants to assess the role of 14-3-3/NMDAR interaction in synaptic targeting of these receptors.
NMDAR hypofunctionality is recognized as one of the pathological mechanisms for SCZ [12,[73][74][75][76][77]. Accumulating evidence supports the idea that NMDAR hypofunctionality may act as a convergence point for symptoms of SCZ [8]. As SCZ is a heterogenous disorder caused by various risk factors, examining the molecular mechanism underlying NMDAR hypofunctionality will help us understand the pathophysiology of the disorder and identify potential therapeutic targets. Based on human genetic analyses, 14-3-3 proteins are a risk factor for SCZ [28,29,31,33]. Recent bioinformatic studies have also placed 14-3-3 proteins in various signaling pathways linked to SCZ including postsynaptic NMDARs [15]. Using mouse models, we and others have previously demonstrated that inhibition of 14-3-3 proteins causes SCZassociated behaviors and changes in synaptic NMDAR levels. Here we demonstrate that disruption of 14-3-3 functions in glutamatergic cortical and hippocampal neurons leads to decreased synaptic localization of GluN1, GluN2A, and GluN2B subunits. Additionally, we identify that 14-3-3 proteins enhance surface delivery of functional GluN2A-and GluN2Bcontaining NMDARs, which may play a critical role in the synaptic localization of these receptors. Further investigation of these signaling pathways may shed light on how dysfunction of 14-3-3 proteins causes NMDAR hypofunctionality and leads to SCZ pathogenesis.